The regulation of stem cell proliferation and differentiation is essential for tissue homeostasis in metazoans and its misregulation causes tissue dysfunction, cancer, or degenerative diseases. The aim of this application is to illustrate the mechanism by which stem cells exit from mitosis and transit into endoreplication in the Drosophila intestinal stem cell (ISC) lineage. Drosophila ISCs display similarities in function and regulation with a variety of mammalian somatic stem cells, thus providing an efficient genetically accessible model system to explore mechanisms regulating stem cell proliferation, differentiation, and regeneration. In the adult fly posterior midgut, ISCs are the only cells undergoing mitotic cell division, generating one ISC and one enteroblast (EBs), which goes on to terminally differentiate into a diploid enteroendocrine cell (EE) or a polyploid enterocyte (EC) EC commitment and differentiation thus requires a regulated transition into endoreplication from mitosis. The precise temporal and spatial regulation of this transition is essential for long-term tissue homeostasis. The applicant's preliminary data suggest that a WD40-repeat family protein, dWdr40A, plays a major role in proliferation and differentiation of the ISC lineage. Genomic and proteomic studies identified the human Wdr40a homologue as a CRL4 (Cullin4-RING E3 ubiquitin ligase) component. Its function, however, is completely unknown. Using lineage tracing approaches the applicant found that loss of dWdr40a increases ISC proliferative activity, while overexpression of dWDR40a significantly inhibits ISC proliferation, suggesting that dWdr40a negatively regulates ISC proliferation. Furthermore, dWdr40a seems to limit the entry of ISC daughter cells into an endoreplicating cell cycle. These phenotypes are similar to phenotypes observed in conditions in which CRL4 function is perturbed, suggesting that dWdr40a influences the transition from a mitotic cell cycle to endoreplication in the ISC lineage by regulating CRL4 activity. It is likely that the transcription factor E2F1 is the mediator of this function, since a recent study suggests that CRL4 governs an oscillating destruction of E2F1 to initiate endoreplication. Accordingly, overexpression of dWDR40a phenocopies the expression of stabilized E2F1 in salivary gland cells. Based on these data, the applicant proposes that dWDR40a regulates the destruction of E2F, a master regulator of the mitosis-to-endoreplication switch, by negatively controlling CRL4 activity. The proposed studies will (1) establish the function of dwdr40a in ISC proliferation and differentiation using genetic approaches to study the interaction between dwdr40a and E2F1, as well as, between dwdr40a and CLR4CDT2 complexes, (2) investigate the physical interaction of WDR40a with CRL4 complexes in Drosophila S2 and mammalian cell culture, and (3) identify novel regulators that control ISC proliferation and differentiation using genetic screens. The proposed study will characterize a novel, evolutionarily conserved, regulatory machinery that controls stem cell proliferation and differentiation, providing new insights into somatic stem cell function and regeneration.